for E. histolytica/E. dispar versus E. coli.
3. Certain organisms [G. lamblia (G. duodenalis, G. intestinalis), hookworm eggs, and occasionally Trichuris eggs] may not concentrate as well from PVA-preserved specimens as they do from those preserved in formalin. However, if enough Giardia organisms are present to concentrate from formalin, PVA should contain enough for detection on the permanent stained smear. In clinically important infections, the number of helminth eggs present would ensure detection regardless of the type of preservative used. Also, the morphology of Strongyloides stercoralis larvae is not as clear from specimens in PVA as from specimens fixed in formalin.
4. For unknown reasons, C. belli oocysts are routinely missed in the concentrate sediment when concentrated from PVA-preserved specimens. The oocysts would be found if the same specimen were preserved in formalin rather than PVA.
5. In past publications, recommended centrifugation times have not taken into account potential problems with the recovery of Cryptosporidium oocysts. There is anecdotal evidence strongly indicating that Cryptosporidium oocysts may be missed unless the centrifugation speed is 500 × g for a minimum of 10 min.
6. Adequate centrifugation time and speed have become very important for recovery of microsporidial spores. In some of the earlier publications, use of uncentrifuged material was recommended. However, we have found that centrifugation for 10 min at 500 × g definitely increases the number of microsporidial spores available for staining and subsequent examination.
Iodine-Trichrome Stain for Sediment
A combination of Lugol’s iodine solution and trichrome stain can be used to stain fecal sediment from the concentration procedure (24). Coloring the eggs and cysts yellow-brown (iodine) and the debris green (trichrome) provides contrast which facilitates the detection of parasites. The use of such an approach usually depends on personal preferences and the results of parallel trials of the current method and new methods being considered. This iodine-trichrome wet examination can be used as an adjunct procedure but does not take the place of the unstained wet examination of the sediment.
Quality Control for Iodine-Trichrome Stain for Sediment
1. Check the working iodine solution each time it is used or periodically (once a week). The iodine and trichrome stain solutions should be free of any signs of bacterial or fungal contamination.
2. The iodine should be the color of strong tea (discard if it is too light).
3. Protozoan cysts stained with iodine should contain yellow-gold cytoplasm, brown glycogen material, and paler refractile nuclei. The chromatoidal bodies may not be as clearly visible as in a saline mount. Human WBCs (buffy coat cells) mixed with negative stool can be used as a QC specimen. The human cells stain with the same color as that seen in the protozoa. The background debris stains green from the components of the trichrome stain.
4. If appropriate due to extensive use and/or lack of routine maintenance, the microscope should be calibrated (within the last 12 months), and the original optics used for the calibration should be in place on the microscope when objects are being measured. The calibration factors for all objectives should be posted on the microscope or close by for easy access.
5. All QC results should be appropriately recorded; the laboratory should also have an action plan for “out-of-control” results.
Procedure for Iodine-Trichrome Stain for Sediment
1. Place 4 drops of Lugol’s iodine solution into a test tube.
2. Place 4 drops of fecal concentrate into the test tube. Mix well.
3. Place 2 drops of the Lugol’s iodine solution-fecal concentrate mixture from step 2 on a glass slide.
4. Add 1 drop of trichrome stain. Mix with a wooden applicator stick, and cover with a coverslip (22 by 22 mm, no. 1).
5. Microscopically examine the entire preparation under low power (×100) and at least one-third to one-half of the area under high dry power (×400).
Results and Patient Reports from Iodine-Trichrome Stain for Sediment
Protozoan trophozoites and/or cysts and some helminth eggs and larvae may be seen and identified. Lugol’s iodine stains A. lumbricoides and Taenia eggs quite dark; these eggs are difficult to recognize and may be mistaken for debris, hence the need for the unstained sediment examination. In a heavy infection with Cryptosporidium spp., oocysts may be seen in a direct smear; however, some type of modified acid-fast stain or fecal immunoassay is normally used to detect these organisms, particularly when few oocysts are present. Oocysts of C. belli can also be seen in a direct smear. Cyclospora oocysts may not be recognized because they resemble debris. Spores of the microsporidia are too small, and the shape resembles that of other debris within the stool; therefore, they are not readily visible in a direct smear.
1. Protozoan cysts may or may not be identified to the species level (depending on the clarity of the morphology).
Example: Iodamoeba bütschlii cysts
2. Helminth eggs and/or larvae may be identified.
Example: Hymenolepis nana eggs
3. C. belli oocysts may be identified; however, Cyclospora and Cryptosporidium oocysts are generally too small to be recognized or identified. Subsequent immunoassays or modified acid-fast staining is recommended.
Example: Cystoisospora (Isospora) belli oocysts
4. Artifacts and/or other structures may also be seen and reported as follows.
Note These cells are quantitated; however, the quantity is usually assessed when the permanent stained smear is examined under oil immersion.
Example: Moderate PMNs
Procedure Notes for Iodine-Trichrome Stain for Sediment
1. As mentioned above, some workers prefer to make the wet mounts on larger slides. Often, there is less chance of getting fluids on the microscope stage if larger slides are used.
2. This stain is darker than the traditional iodine stain. The microscope light should be increased over that used for the unstained wet smear examination.
Procedure Limitations for Iodine-Trichrome Stain for Sediment
1. Because this is a darker stain than routine iodine stains, it is important to also examine a saline wet smear. This is particularly important because A. lumbricoides and Taenia eggs stain too dark with the iodine and may not be recognized as helminth eggs.
2. Results obtained with wet smears should usually be confirmed by permanent stained smears. Some protozoa are very small and difficult to identify to the species level with just the direct wet smears. Confirmation is particularly important for E. histolytica/E. dispar versus E. coli. These findings can be reported as “preliminary report, based on direct wet smear examination only,” and the final report can be submitted after the concentration and permanent stain procedures are completed. However, if the examination turnaround time is approximately 24 h or less, there is no need for a preliminary report; the final report can be submitted after completion of the concentration and permanent stained smear examinations.
Zinc Sulfate Flotation Concentration
The flotation procedure permits the separation of protozoan cysts and eggs of certain helminths
